Abstract

Human memory T cells (TM cells) that produce IL-17 or IL-22 are currently defined as Th17 or Th22 cells, respectively. These T cell lineages are almost exclusively CCR6+ and are important mediators of chronic inflammation and autoimmunity. However, little is known about the mechanisms controlling IL-17/IL-22 expression in memory Th17/Th22 subsets. We show that common γ chain (γc)–using cytokines, namely IL-2, IL-7, and IL-15, potently induce Th17-signature cytokine expression (Il17a, Il17f, Il22, and Il26) in CCR6+, but not CCR6−, TM cells, even in CCR6+ cells lacking IL-17 expression ex vivo. Inhibition of phosphoinositide 3-kinase (PI-3K) or Akt signaling selectively prevents Th17 cytokine induction by γc-cytokines, as does ectopic expression of the transcription factors FOXO1 or KLF2, which are repressed by PI-3K signaling. These results indicate that Th17 cytokines are tuned by PI-3K signaling in CCR6+ TM cells, which may contribute to chronic or autoimmune inflammation. Furthermore, these findings suggest that ex vivo analysis of IL-17 expression may greatly underestimate the frequency and pathogenic potential of the human Th17 compartment.

The IL-2 family of cytokines, which signal through multimeric receptors containing the shared common γ chain (γc) subunit, includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. These cytokines, particularly IL-2, IL-7, and IL-15, play pivotal roles in promoting T cell development, homeostasis, and differentiation. In addition to activating Jak–Stat pathways, γc signaling induces the generation of lipid second messengers through activation of phosphoinositide 3-kinase (PI-3K; Rochman et al., 2009). One of these second messengers, PI(3,4,5)P3 (phosphatidylinositol-(3,4,5)-trisphosphate), binds to the PH (pleckstrin homology) domain of proteins and controls the activity and function of several signaling molecules, including the serine/threonine protein kinase Akt (Fruman, 2004). In turn, Akt directly phosphorylates the transcription factor FOXO1 (Forkhead box protein O1), thereby preventing its nuclear translocation and transcriptional activity (Brunet et al., 1999). Inhibition of FOXO1 by PI-3K has been shown to be essential for γc-cytokine signaling–mediated cell survival, proliferation, and glucose utilization in leukemia cells (Barata et al., 2004). In contrast, activation of FOXO1, by way of reduced PI-3K signaling, can lead to the expression of another transcription factor called KLF2 (kruppel-like factor 2; Sinclair et al., 2008; Kerdiles et al., 2009), which has been implicated in the modulation of IFN-γ and IL-4 production in human and mouse T cells (Weinreich et al., 2009, 2010; Bu et al., 2010). However, whether FOXO1 or KLF2 act downstream of PI-3K to regulate effector cytokine production in human TM cells is not yet known.

In searching for stimuli that regulate the production of IL-17 and IL-22 in human TM cells, we found that γc-cytokines, notably IL-2, IL-7, or IL-15, are sufficient to induce de novo expression of IL-17, IL-22, and other Th17-signature cytokines in CCR6+, but not CCR6−, TM cells. Treatment of cytokine-stimulated CCR6+ TM cells with small molecule inhibitors of PI-3K or Akt repressed γc-cytokine–driven IL-17/IL-22 expression, as did ectopic expression of FOXO1 or KLF2. These findings suggest that PI-3K signaling may amplify Th17- or Th22-associated tissue inflammation by promoting proinflammatory cytokine expression in lineage-committed human Th17/Th22 cells. Our results also demonstrate that the frequency of human TM cells harboring the capacity to secrete IL-17/IL-22 in vivo may be substantially higher than what is predicted based on ex vivo cytokine analysis.

RESULTS

De novo induction of IL-17 in CCR6+ IL-17− T cells is driven by γc-cytokines

Nearly all T cells defined as Th17, by virtue of IL-17 expression, also express CCR6, although only a fraction of CCR6+ T cells (∼5–10%) actively express IL-17 upon ex vivo stimulation. Given that both CCR6 and IL-17 are up-regulated by ectopic expression of RORC in human T cells (Manel et al., 2008) and that CCR6 is a direct target gene of the key Th17 transcription factor Stat3 (Durant et al., 2010), we hypothesized that expression of this chemokine receptor may broadly define human T cells that have the capacity to express IL-17, even if they lack IL-17 expression ex vivo. To address this possibility, we FACS sorted primary human CD4+CD45RO+CD25− TM cells stimulated either with beads conjugated to anti-CD3 and anti-CD28 (aCD3/aCD28) antibodies or with PMA and ionomycin into CCR6−IL-17−, CCR6+IL-17+, or CCR6+IL-17− subsets using a cytokine capture assay (Fig. 1 A and Fig. S1 A). Analyses of gene expression directly after sorting confirmed that Il-17a transcript levels were substantially higher in CCR6+IL-17+ cells compared with either CCR6− or CCR6+IL-17− cells, irrespective of the activating stimuli (i.e., aCD3/aCD28 or PMA and ionomycin). However, both CCR6+ T cell subsets displayed elevated RORC expression levels that ranged between 80- and 400-fold higher than those observed in CCR6− TM cells (Fig. 1 B and Fig. S1 B; Singh et al., 2008). Given that RORC was highly expressed in CCR6+ TM cells independent of ex vivo IL-17 production, we next cultured these T cell subsets in IL-2–supplemented medium for 6 d to ask whether CCR6+IL-17− cells could up-regulate IL-17 expression. As expected, a large majority of cells initially sorted as CCR6+IL-17+ maintained high-level IL-17 expression upon restimulation, whereas CCR6−IL-17− cells remained largely IL-17 negative (Fig. 1 C and Fig. S1 C). Remarkably, 20–40% of the CCR6+ cells initially sorted as IL-17− expressed IL-17 after culture with IL-2 (Fig. 1 C and Fig. S1 C). The appearance of IL-17–producing T cells within ex vivo–isolated CCR6+IL-17− cultures was not a result of selective outgrowth of residual IL-17+ cells, as FACS-sorted CCR6+IL-17− and CCR6+IL17+ cells proliferated equally well in response to IL-2 stimulation, as seen by fluorescent dye dilution and mixed co-culture experiments (Fig. S1, D–G).

De novo expression of IL-17 in CCR6+IL-17− human TM cells is induced by γc-cytokines. (A) Total CD4+ TM cells (CD45RO+CD25−) were stimulated for 18–24 h with aCD3/aCD28 beads and then were FACS sorted into CCR6−IL-17−, CCR6+IL-17−, or CCR6+IL-17+ populations by cytokine capture assay. (B) Cells sorted as in A after aCD3/aCD28 stimulation were analyzed by quantitative (q) PCR for expression of Il-17a and Rorc gene expression. Data are presented as the mean fold change from duplicates from one representative donor and are derived from Ct values (qPCR) ± SD from duplicate samples. The data were obtained from a single donor that is representative of two to three independent experiments using T cells from different donors. (C) Live CCR6−IL-17−, CCR6+IL-17−, or CCR6+IL-17+ T cells were maintained in IL-2–containing medium for 6–7 d and were restimulated with PMA and ionomycin. Cytokine production was determined by intracellular staining. (D) Cytokine gene expression was determined using qNPA (see Materials and methods) in CCR6−IL17−, CCR6+IL-17−, or CCR6+IL-17+ populations expanded for 7 d in IL-2–supplemented medium and restimulated with PMA and ionomycin. Cytokine mRNA expression was normalized to a housekeeping gene (Rpl19) and normalized values are presented as a fold change on a scatter plot (where x axis = CCR6+IL-17−/CCR6−IL-17− and y axis = CCR6+IL-17+/CCR6−IL-17−). Data from A–D is representative of three independent donors. (E) FACS-sorted CCR6−IL-17− cells (white bars), or CCR6+IL-17− cells (black bars) sorted as in A, were cultured in different cytokine conditions as indicated for 6 d. IL-17 expression was determined by FACS analysis after restimulation with PMA and ionomycin. Data are presented as the mean percentage of cells ± SD from three different donors. (F) CCR6+IL-17− TM cells are specifically poised to express IL-17 in human RA patients. Total CD4+ TM cells isolated from the peripheral blood of RA patients were either stimulated directly ex vivo (day 0) with PMA and ionomycin or were stimulated and FACS sorted as in A into CCR6−IL-17− or CCR6+IL-17− cells. These cells were cultured in IL-2–supplemented medium for 7 d before restimulation with PMA and ionomycin (day 7). The frequencies of IL-17–producing cells were determined via intracellular staining and FACS analysis. Data represent the mean (horizontal bars) percentage of IL-17-producing cells from four individual RA patients.

A more comprehensive analysis of cytokine gene expression by human CCR6+IL-17− and CCR6+IL-17+ cells revealed that these two cell types were nearly indistinguishable after culture with IL-2. Specifically, several proinflammatory cytokines canonically associated with the Th17 lineage (IL-17a, IL-17f, IL-22, IL-26, Ccl20, and IL-21) were comparably expressed in both CCR6+IL-17− and CCR6+IL-17+ cells, although both CCR6+ cell types expressed substantially higher levels of these cytokines compared with CCR6− TM cells (Fig. 1 D). Importantly, differential expression of Th17-signature cytokines by both CCR6+ populations compared with CCR6− T cells was specific, as all three populations expressed roughly equivalent levels of other cytokines not related to the Th17 lineage, namely Tnf, Il4, and Ifng (Fig. 1 D).

Given that IL-2 is the prototype of the IL-2 family of cytokines, all of which signal through cytokine receptors comprised in part by the γc subunit, we next asked whether other γc-cytokines could also induce de novo IL-17 production by CCR6+IL-17− TM cells. Both IL-7 and IL-15 induced similar levels of IL-17 expression in CCR6+IL-17− T cells, whereas IL-23, which is known to enhance Th17 cell differentiation (Ivanov et al., 2007), did not influence IL-17 expression in the absence of IL-2 (Fig. 1 E). In contrast, and as observed for IL-2, neither IL-7 nor IL-15 induced IL-17 expression in CCR6− TM cells. In addition, CCR6+, but not CCR6−, IL-17− TM cells isolated from peripheral lymphoid organs of wild-type C57B/6 mice were capable of producing IL-17 after 6 d in culture with IL-2 (Fig. S2).

These findings suggest that ex vivo analyses of IL-17 expression underestimate the frequency of TM cells that can express IL-17 in inflammatory settings. Because several studies have investigated changes in Th17 frequencies within autoimmune patient cohorts, we asked whether CCR6+IL-17− TM cells isolated from the peripheral blood of patients with RA could be similarly induced to express IL-17 by IL-2 stimulation. Indeed, we observed that CCR6+, but not CCR6−, IL-17− TM cells from RA patients up-regulated IL-17 after culture with IL-2 to similar levels as those observed in healthy adult donors (Fig. 1 F). Collectively, these data demonstrate that CCR6+ TM cells are uniquely poised to express IL-17 in response to IL-2 stimulation irrespective of their IL-17 phenotype ex vivo. These results also indicate that this inflammatory feature of CCR6+ TM cells is conserved between humans and mice.

Human CCR6+ Th17 cells have been reported to be enriched within CXCR3− or CD161+ subsets as cells of these subphenotypes produce more IL-17 upon ex vivo stimulation compared with either CCR6+CXCR3+ or CCR6+CD161− cells (Acosta-Rodriguez et al., 2007; Cosmi et al., 2008). We confirmed that a significant proportion of human CCR6+ TM cells coexpressed either CD161 or CXCR3 and that CD161 expression was enriched within CCR6+ cells, whereas expression of CXCR3 was enriched within the CCR6− compartment (Fig. 2 A). As expected, CCR6+CD161+ TM cells were enriched for IL-17–producing cells upon ex vivo stimulation compared with CCR6+CD161− cells, whereas CCR6+CXCR3+ cells produced less IL-17 but more IFN-γ than CCR6+CXCR3− cells (Fig. 2 A). To address the possibility that IL-17 up-regulation by CCR6+IL-17− cells is restricted to predefined CCR6+ subpopulations, we fractionated CCR6+IL-17− cells based on either CD161 or CXCR3 expression and expanded these subsets in the presence of IL-2 (Fig. 2 A). After expansion and restimulation, we found that all CCR6+IL-17− subsets were capable of high-level IL-17 production in contrast to CCR6− TM cells (Fig. 2, B and C). Consistent with the ex vivo analyses of these subsets, however, the frequency of IL-17+ cells was somewhat higher (approximately twofold) in the CD161+ or CXCR3− subsets compared with CD161− or CXCR3+ counterparts, respectively (Fig. 2, B and C). Moreover, CCR6+CXCR3+IL-17− cells maintained their propensity to express IFN-γ as compared with CCR6+CXCR3− memory cells (Fig. 2 C). These data demonstrate that heterogeneous subpopulations of CCR6+ T cells share a capacity to express IL-17 in response to γc-cytokine stimulation.

IL-17 induction in response to γc-cytokine stimulation is a conserved feature shared by heterogeneous CCR6+ TM cell subsets. (A) Ex vivo (day 0) isolated CD4+ TM cells were costained with CCR6, CD161, and CXCR3. The frequency of CCR6+ cells positive or negative for either CD161 (left) or CXCR3 (right) are shown. Ex vivo cytokine production was determined after PMA and ionomycin stimulation within each gated CCR6+ TM cell subset as indicated. FACS plots show the intracellular expression of IL-17, IL-22, and IFN-γ. (B) FACS-sorted CCR6−IL-17−, CCR6+CD161+/−IL-17− (left), or CCR6+CXCR3+/−IL-17− (right) cells were cultured in IL-2–supplemented medium for 7 d and restimulated with PMA and ionomycin. Cytokine production was determined by intracellular staining for IL-17, IL-22, and IFN-γ. Each set of data is representative of three donors.

PI-3K/Akt signaling mediates Th17 cytokine induction by γc-cytokines

We have previously shown that γc-cytokines, such as IL-7, can promote T cell survival in the absence of TCR signaling and do so without inducing significant cell division (Unutmaz et al., 1994, 1999). Given that IL-7 and IL-15 could substitute for IL-2 in inducing Th17 cytokine expression in TCR-activated CCR6+ TM cells, we next asked whether γc-cytokine signaling was sufficient to promote IL-17/IL-22 expression in resting human CCR6+ TM cells not stimulated through the TCR (Fig. 3 A). Indeed, stimulation of resting CCR6+CD45RO+CD25− TM cells with IL-7 for 10 d consistently enhanced the expression of both IL-17 and IL-22 more than threefold compared with the same cells isolated ex vivo (day 0; Fig. 3, B and C). Importantly, IL-7–mediated induction of IL-17 and IL-22 expression was restricted to CCR6+ cells, as CCR6− TM cells stimulated with IL-7 failed to express IL-17 or IL-22 (Fig. 3 B). Moreover, culture of resting CCR6+ or CCR6− TM cells with IL-7 had almost no impact on IFN-γ production (Fig. 3, B and C). Stimulation of resting CCR6+ TM cells with IL-15 led to similar increases in IL-17/IL-22 expression (unpublished data), whereas another γc-cytokine, IL-4, did not promote Th17 cytokine production in resting CCR6+ TM cells or confer TM cell survival (Fig. S3 B and not depicted). Interestingly, IL-23 did not significantly synergize with IL-7 to further induce IL-17 or IL-22 expression (Fig. S3 B).

PI-3K/Akt inhibitors suppress IL-17 and IL-22 production from IL-7–stimulated CCR6+ TM cells. (A) Resting CD4+CD45RO+CD25−CCR6+/− T cells were cultured in IL-7–containing medium for 7 d followed by culture with or without PI-3K signaling inhibitors for an extra 2 d. Cells were then stimulated with PMA and ionomycin and subjected to intracellular staining and FACS analysis as described in Materials and methods. (B) Blockade of PI-3K signaling suppresses IL-17 and IL-22 production induced by IL-7 from CCR6+ TM cells. Intracellular cytokine staining was performed on CCR6+ and CCR6− TM cells ex vivo (day 0) and after different culturing conditions (day 10) as described in A. CCR6+/− TM cells were cultured with IL-7 alone or plus 5 µg/ml LY294002 or 5 µg/ml AKTi-1/2. (C) Frequency of IL-17–producing T cells is increased by γc-cytokine stimulation across different donors. CCR6+ TM cells isolated from seven healthy donors (D) were stimulated either ex vivo or at day 10 after culture with IL-7. (D and E) PI-3K and Akt inhibitors inhibit IL-17, IL-22, and IL-4 expression in IL-7–activated CCR6+ TM cells in a dose-dependent manner. Cells cultured in IL-7 with or without LY294002 or AKTi-1/2 at different concentrations (µg/ml) were collected and analyzed. The frequency of single (D) or double (E) cytokine-producing cells without inhibitors treatment (control, 0) was normalized to 100%. Cytokine percentage of control was calculated accordingly. Data are presented as the mean percentage cytokine ± SD from at least three different donors. (F) Cytokine gene expression in CCR6+/CCR6− TM cells. Different cytokine gene expression was analyzed by qNPA from either ex vivo freshly isolated CCR6+ (filled rectangle) or CCR6− (empty rectangle) cells at day 0 (d0) and day 10 (d10) after culturing in IL-7–containing medium in the absence or presence of 5 µg/ml PI-3K/Akt inhibitors. Data from CCR6+ TM cells are presented as mean mRNA abundance (AU) ± SD from three different donors. The statistical analyses, either ex vivo or at day 10 with PI-3K inhibitor treatments, are relative to day 10 IL-7 only (filled blue rectangle). Data from CCR6− is representative out of three donors. *, P < 0.05; **, P < 0.005; ***, P < 0.001. NS, no significance.

Human CCR6+ T cells can express IL-17 alone or in combination with other proinflammatory cytokines such as IL-22 or IFN-γ. Cells coexpressing IFN-γ and IL-17 have been shown to share functional characteristics with those expressing IL-17 alone (Boniface et al., 2010), whereas the function of IL-17+IL-22+ cells is unclear. IL-7 stimulation had even more profound effects on the proportion of cells that coexpressed either IL-22 or IFN-γ with IL-17; these double cytokine-producing cells increased more than fivefold in response to IL-7 signaling (Fig. 3 B). Together, these results demonstrate that γc-cytokines, particularly IL-2, IL-7, and IL-15 potently enhance IL-17/IL-22 secretion by CCR6+ TM cell subsets.

Signaling through γc-containing cytokine receptors regulates T cell survival and homeostasis through activation of the PI-3K–Akt pathway (Rochman et al., 2009). To determine if this pathway also controls expression of IL-17 and IL-22, we treated CCR6+ TM cells that had been cultured for 1 wk in IL-7 with or without the PI-3K inhibitor LY294002 or the Akt inhibitor AKTi-1/2 for additional 48 h (Fig. 3 A). We confirmed that treatment of Jurkat cells with 1–10 µg/ml of either inhibitor down-regulated cytokine-dependent phosphorylation of Akt without adversely affecting cell viability (Fig. S3 A). Control- or compound-treated T cells were then restimulated to analyze their impact on cytokine production. Treatment with either LY294002 or AKTi-1/2 repressed γc-mediated IL-17/IL-22 induction in a dose-dependent manner, whereas expression of IL-4 or IFN-γ was either less reduced or not affected, respectively (Fig. 3, B, D, and E). Inhibitory effects of the PI-3K and Akt inhibitors were even more profound when considering the proportion of T cells coexpressing IL-22 or IFN-γ together with IL-17 (Fig. 3 E). Furthermore, treatment with PI-3K or Akt inhibitors also repressed IL-17 and IL-22 expression in TCR-activated CCR6+ TM cells cultured in IL-2 (Fig. S3, C–E). These effects were reversible because IL-17/IL-22 levels were restored upon removal of PI-3K or Akt inhibitors from culture medium (unpublished data). Moreover, IL-17/IL-22 repression by PI-3K or Akt inhibition was not associated with changes in Rorc or Foxp3 expression levels (Fig. S3 F).

We next evaluated the impact of PI-3K or Akt inhibition on the expression of a broader panel of both Th17-related and unrelated cytokines to determine how selectively this pathway regulates inflammatory cytokine expression. In addition to IL-17A, IL-17F, and IL-22, Th17 cells have been shown to preferentially express IL-21 (Nurieva et al., 2007), IL-26 (in human cells), CCL20 (Wilson et al., 2007), and, in some instances, IL-10 (Zhu and Paul, 2010). Although intracellular IL-10 production was not detectable in either CCR6+ or CCR6− TM cells upon stimulation ex vivo or after culture with IL-7, we found that, unlike IL-17 and IL-22, ex vivo production of IL-21 was equivalent in both human CCR6− and CCR6+ TM cell subsets (Fig. S4, A and B). Similarly, and in contrast to IL-17 and IL-22, expression of IL-21 was not increased in either CCR6+ or CCR6− TM cells upon stimulation with IL-7, and in fact was slightly reduced (Fig. 3 F; and Fig. S4, A and B). IL-21 expression was, however, sensitive to treatment with PI-3K or Akt inhibitors (Fig. S4, A and B). Ccl20 was preferentially expressed ex vivo by CCR6+ TM cells (Fig. 1 D) and was neither significantly induced by IL-7 stimulation nor repressed by PI-3K pathway inhibition (Fig. 3 F and Fig. S4 C). The expression of other non–Th17-related cytokines, such as Il2, Ifng, Tnf, and Tgfb1 were not influenced by IL-7 stimulation or by treatment with PI-3K or Akt inhibitors (Fig. 3 F and Fig. S4 C). Thus, PI-3K signaling downstream of γc-cytokine receptors promotes the expression of Th17-signature cytokines (Il-17a, Il-17f, and Il-22) within CCR6+ TM cells and does so with a high degree of specificity over other unrelated cytokines.

FOXO1 and KLF2 repress IL-17/IL-22 expression

PI-3K activation downstream of γc-cytokine stimulation negatively regulates FOXO1 nuclear localization and transcriptional activity (Birkenkamp and Coffer, 2003). Because we found cytokine-driven IL-17 and IL-22 expression by CCR6+ TM cells to be sensitive to PI-3K or Akt inhibition, we next asked whether ectopic expression of FOXO1 could also repress γc-mediated IL-17/IL-22 expression. Human CCR6+ TM cells were activated and transduced with control (empty vector [EV]) or FOXO1-expressing lentiviruses and then expanded in the presence of IL-2, FOXO1 overexpression was confirmed by intracellular staining (Fig. S5 A), and these cells were then restimulated to evaluate effects on cytokine production. Analogous to treatment of cells with PI-3K and Akt inhibitors, FOXO1 overexpression reduced both IL-17 and IL-22 secretion (Fig. 4, A and B).

IL-17/IL-22 production from CCR6+ TM cells is suppressed by ectopic FOXO1 expression. (A) T cells activated through their TCR were transduced with FOXO1-GFP and cultured in IL-2 for 14 d then stimulated with PMA and ionomycin, stained for intracellular cytokines, and subjected to FACS analysis. (B) The frequency of single or double cytokine-producing cells from FOXO1 or EV-transduced cells is shown. The horizontal bars are presented as mean frequency of cytokine producing cells from four independent individuals. *, P < 0.05. NS, no significance.

Klf2 expression is regulated by γc-cytokine–induced PI-3K signaling pathway. (A) Klf2 message is abundant in resting CD4+ T cells and down-regulated upon activation through the TCR. Total cellular RNA was extracted from cell pellets of resting aCD3/aCD28 bead–activated CD4+ TM cells. The RNA was subjected to RT-qPCR for Klf2 and b-actin mRNA quantification. (B) Blockade of PI-3K signaling by LY294002 treatment up-regulates Klf2 transcripts in a dose-dependent manner in CCR6+ TM cells cultured in IL-7. CCR6+ TM cells were cultured in IL-7–containing medium as in Fig. 3 A. Total RNA from either ex vivo freshly isolated (day 0) or cultured in IL-7 (day 10) with or without LY294002 at the indicated concentrations was collected and subjected to RT-qPCR to detect Klf2 levels. (C) Ectopic transduction of KLF2 in TCR-activated IL-2–cultured TM cells maintains Klf2 message levels comparable to resting cells. Data shown is from one representative of three different donors.

KLF2 suppresses IL-17 and IL-22 production from CCR6+ TM cells in the presence of ectopic RORC expression. (A and B) KLF2- or shKLF2-transduced CCR6+ TM cells cultured for 2 wk in IL-2 medium were treated with PMA and ionomycin and then subjected to intracellular IL-17, IL-22, IFN-γ, and IL-4 staining. shCont., control shRNA; shKLF2, KLF2 shRNA. The frequency of single or double cytokine-producing cells from either EV- or control shRNA–transduced cells was normalized to 100%. (C) RORC overexpression and silencing of KLF2 are synergistic on induction of IL-17. CCR6− TM cells co-transduced with shKLF2- and RORC-expressing lentiviruses or their corresponding controls were restimulated with PMA and ionomycin for 5 h. Cells were then stained with antibodies against IL-17, IL-22, IFN-γ, and IL-4 and shown as dot plots in C from a representative donor or, graphically, from multiple donors in D. Data are presented as mean percentage of cytokine ± SD from at least three individual donors and independent experiments. *, P < 0.05; **, P < 0.005. NS, no significance.

DISCUSSION

In this paper, we show that Th17-signature cytokines, particularly IL-17 and IL-22, are dynamically regulated within human CCR6+ TM cells by γc-cytokine signals. IL-17 and IL-22 expression by CCR6+ TM cells is controlled by PI-3K/Akt-dependent signaling that involves the repression of both FOXO1 and KLF2. We also report that CCR6 expression on nonregulatory (i.e., CD25−) TM cells broadly defines lineage-committed Th17 cells; phenotypically diverse subpopulations of CCR6+ (e.g., CD161+/−, CXCR3+/−) TM cells all share a capacity to express Th17 cytokines in response to γc-cytokine stimulation, irrespective of whether or not they express IL-17 ex vivo. Importantly, we show that the specific conversion of ex vivo–isolated CCR6+, but not CCR6−IL-17−, TM cells is conserved in mice (Fig. S2). Collectively, these results suggest that IL-17 and IL-22 production by CCR6+ TM cells may be influenced in a bystander fashion by γc-cytokines present at sites of inflammation or infection.

These results also suggest that current methods of calculating Th17 cell frequencies based solely on ex vivo IL-17 production may not represent the true size or inflammatory potential of the human Th17 memory cell compartment. This becomes particularly important for how Th17 cells are evaluated and quantified in clinical disease settings. Indeed, we have found that a substantial portion of CCR6+, but not CCR6−, IL-17− TM cells isolated ex vivo from the peripheral blood of RA patients convert into IL-17 producers upon culture with γc-cytokines (Fig. 1 F).

The ability of CCR6+ TM cells to express IL-17 may be a result of the fact that, in contrast to CCR6− TM cells, CCR6+ cells express high levels of RORC ex vivo, independent of IL-17. This is indeed likely given that ectopic expression of RORC can induce IL-17 expression in CCR6− TM cells (Fig. 6; Manel et al., 2008). These findings further suggest that RORC is necessary, but not sufficient, for IL-17 production by human TM cells. We propose that RORC synergizes with γc-mediated PI-3K and Akt signaling to trans-activate IL-17 expression. Two additional results support a model in which γc-cytokine signaling acts together with RORC to control IL-17 production by itself, as opposed to regulating Th17 lineage commitment. First, IL-7 stimulation, treatment with PI-3K or Akt inhibitors, and overexpression of FOXO1 or KLF2 did not affect RORC mRNA expression levels (Fig. S3 F and not depicted). Second, inhibition of IL-17 and IL-22 expression by treatment with LY294002 was completely reversible (unpublished data).

IL-23 is another cytokine thought to be important for the stability and proinflammatory functions of Th17 cells, particularly in the mouse (Zheng et al., 2007; Zhou et al., 2007). However, we found that IL-23 was not able to compensate for γc-cytokines in the induction of IL-17/IL-22 expression from CCR6+ human TM cells, even though these cells expressed Il23R mRNA (unpublished data). Conceivably, IL-23 could enhance IL-17–secreting cells but this effect may be attributed to preferential expansion of CCR6+IL-23R+ TM cells that are more likely to express IL-17 upon TCR triggering. It is also reasonable to speculate that a more dominant role of IL-23 in driving IL-17–associated pathology is through its reported effects on IL-17 expression within TCR-γ/δ lymphocytes or other innate immune cells (Moens et al., 2011).

PI-3K is activated in T cells in response to TCR engagement or γc-cytokine stimulation (Rochman et al., 2009; Smith-Garvin et al., 2009), which in turn results in the phosphorylation and activation of Akt (Coffer et al., 1998). On one hand, PI-3K/Akt signaling positively regulates cell metabolism through activation of mTORC1 (Gibbons et al., 2009). On the other hand, activated Akt also inhibits the transcriptional activity or expression of FOXO1 or KLF2, respectively (Fabre et al., 2008; Sinclair et al., 2008). We found that the induction of IL-17 and IL-22 expression by γc-cytokines was reversed by short-term (48 h) treatment with PI-3K or Akt inhibitors. Furthermore, overexpression of either FOXO1 or KLF2 recapitulated the effects of the PI-3K or Akt inhibitors on IL-17 and IL-22 induction. Conversely, shRNA-mediated silencing of endogenous KLF2 enhanced IL-17 and IL-22 expression by CCR6+ TM or RORC-expressing CCR6− TM cells. We therefore conclude that γc-dependent activation of PI-3K and Akt drives expression of IL-17 and IL-22, preferentially through a pathway that involves FOXO1 and KLF2 down-modulation (Fig. S6; Brunet et al., 1999; Barata et al., 2004; Fabre et al., 2005, 2008; Sinclair et al., 2008). However, as rapamycin treatment (Kopf et al., 2007) and mTOR deficiency (Delgoffe et al., 2009, 2011) have been reported to negatively regulate Th17 differentiation in naive mouse T cells, it will be of interest to determine whether mTOR signaling also controls Th17-signature cytokine production in human CCR6+ TM cells.

The mechanism by which FOXO1 and KLF2 negatively regulate IL-17 and IL-22 expression in human TM cells is unclear. The most obvious possibility is that both FOXO1 and KLF2 function nonredundantly as transcriptional repressors of IL-17 and IL-22 (Fig. S6). However, it is also plausible that FOXO1 regulates IL-17/IL-22 expression indirectly by inducing KLF2 expression. Indeed, previous studies have shown that genetic ablation of Foxo1 leads to down-regulation of Klf2 expression (Fabre et al., 2008; Sinclair et al., 2008; Kerdiles et al., 2009). Consistent with these studies, we found that lentiviral overexpression of FOXO1 in human TM cells led to increased Klf2 expression (unpublished data). It will be important in future studies to determine the mechanism by which these and other transcription factors acutely regulate IL-17/IL-22 expression in human CCR6+ TM cells.

In conclusion, our data indicate that human CCR6+ TM cells represent a broad proinflammatory effector lineage that expresses IL-17 and IL-22 in a dynamic manner relying on synergy between γc-cytokine signaling and RORC. Local immune responses and subsequent accumulation of γc-cytokines may thus contribute to Th17/Th22-driven inflammation, which may be sensitive to PI-3K- or Akt-targeted therapies. Moreover, our findings strongly suggest that ex vivo analyses of IL-17 and IL-22 secretion may vastly underestimate the true frequency and proinflammatory potential of Th17/Th22 cells in vivo. These results shed new light on the regulation of proinflammatory cytokine production by human TM cells and also call for a broader phenotypic definition of Th17/Th22 subsets within both healthy human donors and autoimmune patient populations.

IL-17 secretion assay.

Manufacturer’s protocols (Miltenyi Biotec) for labeling human and mouse IL-17–secreting cells were followed. In brief, activated T cells were washed in cold buffer (PBS + 0.5% BSA + 2 mM EDTA) and labeled with IL-17 catch reagent for 5 min on ice. Prewarmed medium was then added and the cells were incubated at 37°C on a shaker for 45 min. For human IL-17 capture experiments, cells were washed once more with cold buffer and PE-conjugated IL-17 detection antibodies were added and incubated on ice for 10 min. For mouse IL-17 capture experiments, all steps were identical, except that the IL-17 detection antibody used was biotinylated, and a secondary staining step using APC-conjugated streptavidin was used. Cells were then washed and stained with additional cell surface antibodies at room temperature for 20 min. These cells were then washed once more with cold buffer, resuspended in serum-free RPMI 1640 medium, filtered, and FACS sorted. Antibodies used for human IL-17 secretion sorts were CCR6-PercpCy5.5 (cloneTG7/CCR6; BioLegend) and CD25–Alexa Fluor 700 (clone BC96; BioLegend). Antibodies for mouse IL-17 secretion sorts were CD4–Pacific orange (Invitrogen), CD62L–Pacific blue (eBioscience), CCR6-PE (BioLegend), IL-17A–biotin (Miltenyi Biotec), and streptavidin-APC (eBioscience).

Plasmids and gene cloning.

Full-length human KLF2 cDNA (NM_016270; pCMV6-XL4-KLF2; Origene) was cloned into a lentiviral vector at EcoRI sites. This vector consists of HIV-derived vector (HDV), an internal ribosome entry site (IRES), and a gene for RFP. The primers for KLF2 amplification were: 5′ forward, 5′-CCGGAATTCG­CCATGGCGCTG­AGTGAACCCATC-3′; and 3′ reverse, 5′-CCGGAATTCC­TACATGTGCCG­TTTCATGTGCAG-3′. Wild-type FOXO1 (FOXO1-GFP) was a gift from T.G. Unterman (University of Illinois, Chicago, IL). FOXO1-GFP fusion protein was engineered into HDV in frame with XbaI and XhoI sites. The primers for FOXO1 amplification were: 5′ forward, 5′-GCTCTAGAGC­ATTGCCATGGC­CGAGGCGCCT-3′; and 3′ reverse, 5′-TTCTCGAGGCTTACTTGTACAGCTCGTC-3′. The lentivirus-encoding RORC gene, RORC-IRES-HSA, was a gift from D. Littman (Skirball Center, New York University School of Medicine, NY, NY). Short hairpin RNA against KLF2 (shKLF2; Invitrogen) was engineered into a lentiviral vector, as previously described (Antons et al., 2008), targeting position 922 in the cDNA: 5′-GGCAAGACCTACACCAAGAGT-3′.

Lentiviruses production and transductions.

The lentiviruses pseudotyped with VSV-G envelope were generated as previously described (Unutmaz et al., 1999). All lentiviruses expressed GFP, RFP, or heat stable antigen (HSA) as the marker in place of the nef gene, as previously described (Unutmaz et al., 1999; Oswald-Richter et al., 2004, 2007). Viral titers were measured as described (Unutmaz et al., 1999; Oswald-Richter et al., 2007) and ranged from 1–30 × 106 IFU/ml. Activated TM cells were transduced with different genes, such as FOXO1, KLF2, and RORC, or KLF2 shRNA and maintained in IL-2 containing 10% FCS RPMI 1640 medium for 14 d before reactivation with PMA and ionomycin.

RNA isolation and quantitative PCR.

105 purified T cells were snap frozen in liquid nitrogen for total RNA isolation. Total RNA was isolated using RNeasy isolation kit (QIAGEN) according to the manufacturer’s protocol. The extracted RNA was then reverse transcribed into cDNA and quantified using TaqMan Cell to CT kit (Applied Biosystems). The following TaqMan primers and probe sets were purchased from Applied Biosystems: human KLF2 (Hs00360439_g1), RORC (Hs01076120_g1), Il-17a (Hs00174383_m1), b-Actin (Hs03023880_g1), and Foxp3 (Hs00203958_m1). Data were acquired and analyzed on an ABI 7500 sequence detection system. Quantitative real-time PCR data were analyzed by normalizing Ct values of genes of interest to the Ct values of the housekeeping gene b-actin.

Acknowledgments

This work was supported by National Institutes of Health grants R21AI087973 and R01AI065303.

Disclosure: R. Ramesh, I.M. Djuretic, T.J. Carlson, and M.S. Sundrud are affiliated with a commercial organization, Tempero pharmaceuticals, which also provided financial support to the laboratory of D. Unutmaz at New York University. The authors have no conflicting or competing financial interests.

Footnotes

Abbreviations used:

EV

empty vector

PI-3K

phosphoinositide 3-kinase

RA

rheumatoid arthritis

qNPA

quantitative nuclease protection assay

Submitted: 3 December 2010

Accepted: 30 June 2011

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